This invention relates to and has among its objects the isolation, selection, and application of bacteria to control in the field diseases caused by Gaeumannomyces graminis in plants, such as take-all in wheat and Ophiobolus patch in turf grass. Further objects of the invention will be evident from the following description wherein in parts and percentages are by weight unless otherwise specified.
Widespread diseases of cereal crops and turf grass are caused by the soil-borne fungus Gaeumannomyces graminis (Gg) and result in significant economic loss due to reduction in crop yield. Take-all, a disease caused by Gaeumannomyces graminis var tritici (Ggt) is a severe disease of wheat. Ggt also infects other cereal crops such as barley, rye, and oats as well as wild and cultivated grasses. Symptoms of wheat take-all include dark longitudinal lesions on roots; in severe cases, the entire root may become blackened with disease with the fungus migrating to the crown of the wheat plant (where the crown roots originate) and the tillers (stems). Severely infected wheat plants are identified in the field by their white heads which result when infection of the crown by the fungus cuts off water transport to upper plant parts causing the plant to die prematurely. It has been estimated that in the Pacific Northwest (Washington, Oregon, and Idaho), an area where wheat is the fourth most important irrigated crop, take-all commonly causes a 5-20 percent reduction in the yield of wheat. On a world-wide basis, take-all is the most important root disease of wheat, causing reduction in yield in fields where wheat has been grown two or more years in succession.
Another Gg fungus, Gaeumannomyces graminis var. avenae (Gga), infects oats and grasses and has been identified as causing Ophiobolus patch in turf grasses such as bent grass and the like. Gaeumannomyces graminis var graminis (Ggg) infects some grasses and has been suggested as causing Brown Sheath Rot in rice.
Control of Gg-caused disease is important to prevent crop losses and maintain healthy turf grass. Presently, however, control of wheat take-all by fungicides is considered economically impractical and Gg-resistant cereal or grass varieties are not known in spite of searches over the past 50 years.
Some natural suppression of Ggt has been found to occur in certain circumstances. For example, take-all decline (TAD), a natural suppression of take-all, develops in soils where Ggt-susceptible cereals have been grown in monoculture for many years. TAD has been extensively studied in an attempt to determine what conditions are responsible for natural suppression. Theories put forward to explain this phenomenon include changes in the microbiological status of the soil, build-up of antagonistic bacteria, changes in the pathogenicity and population of the fungus, concentration and form of nitrogen in the soil, presence of protective fungi and presence of volatile substances such as ethylene in the soil, D. Hornby in "Take-All Decline: A Theorist's Paradise," Soil-borne Plant Pathogens, Ed. B. Schippers and W. Gams, Academic Press, New York (1979) pp 133-156. Hornby reviewed these explanations and concluded that no single hypothesis could explain take-all decline and that opposing views may be concerned with various facets of the same complex phenomenon.
Investigators have carried out two types of studies to assess microbial antagonism in the suppression of Ggt. One type of study involves the transfer of suppressiveness by incorporating a small amount of monoculture wheat-field soil into a take-all conducive soil. This procedure has had only partial success. For example, when fumigated soil containing added Ggt-inoculum was amended with one percent take-all suppressive soil, restoration of antagonistic properties was provided in the greenhouse; however, the same treatment in field plots resulted in only a dealy of take-all in the first year and suppression in the second year. Plots amended with soil from take-all conducive virgin (uncropped) sites did not show take-all suppression until the third year thus demonstrating the difference in antagonism between cropped and virgin soils, and the transmissibility of a biological factor antagonistic to Ggt in the greenhouse and the field (P. J. Shipton et al., Phytopathology, Volume 63, pp 511-517 (1973) (Shipton et al.)). In a similar study, 10 percent of a take-all suppressive soil from a field cropped 21 consecutive years to wheat and a take-all conducive soil were added to fumigated soil, the mixtures amended with one percent Ggt-inoculum and planted to wheat. After two successively croppings, plants grown in the suppressive soil showed suppression of take-all, while plants grown in the conducive soil were not protected. The roots of plants grown in the suppressive soil had higher numbers of pseudomonads than plants grown in the conducive soil, (Weller and Cook, Phytopathology, Volume 71, p. 264 (1981)). These studies indicate that although suppressiveness can be transferred, suppression of take-all does not occur in the field until after the first or second crop year. In addition to not being completely successful, the method of incorporation of monoculture soil to suppress take-all is impractical on a commercial basis as it requires the transfer of tons of soil to the field plots.
The second type of study of microbial antagonism involves the attempt to identify specific Ggt-antagonistic microorganisms and transfer these organisms to soil to reproduce suppression. Studies of specific Ggt-antagonistic microflora which developed in TAD showed that actinomycetes, fungi, and bacteria, especially Pseudomonas spp., were found prominent at times (Hornby, p. 151). However, not all organisms present in take-all suppressive soil were found to suppress take-all. In field trials, only one percent of bacteria isolated from TAD soil and added back to take-all conducive soil effectively antagonized Ggt (Hornby, p. 142). Shipton et al. developed a pot assay to assess take-all suppression by specific microorganisms in the greenhouse. Using this test, Cook and Rovira (Soil Biology and Biochemistry, Volume 8, pp. 269-273, 1976) (Cook and Rovira) took candidate isolates of bacteria and actimomyces from soil, and from diseased and protected wheat roots and tested them as soil treatments to suppress Ggt. Pure cultures of each isolate were grown for 1-2 days in sterile soil and then this "soil inoculum" was mixed with potting soil (1 g soil inoculum per 100 g potting soil). The soil mixture was infested with the take-all fungus (0.1 to 0.5 percent (w/w) Ggt. oat-kernel per soil mixture) and planted to wheat. Of the isolates tested only eight cultures suppressed take-all in the greenhouse. These were identified as Pseudomonas spp. (seven were fluoroescent). While this work identified bacteria present in TAD soil which could impart suppression to wheat seedlings planted in potting soil in the greenhouse, no practical treatment for control of take-all in the field was demonstrated or suggested by the researchers. An equivalent field treatment by the above method of Cook and Rovira would require about 10 tons of the soil inoculum per acre mixed 6 inches deep.
Another complication in finding a biological control of Ggt was that other experiments, namely, cereal sequence experiments taught away from the use of fluorescent pseudomonads to suppress take-all. These studies indicated that fluorescent pseudomonads were often only a small fraction of the total bacterial antagonists which inhibit Ggt and play little or no role in natural suppression of take-all associated with take-all decline. (Soil Biology and Biochemistry, Volume 13, pp 285-291 (1981) (Brown)). Thus, although some information about the influence of soil microflora on TAD existed, the problem remained of how to screen microorganisms for antagonism to Gg-fungus, to select those which would provide disease suppression under field conditions and to find a practical method of field application. This step from successful antagonism in the greenhouse to success in the field is difficult to achieve because in the greenhouse, conditions such as soil temperature, soil moisture, other plant disease, and the like are controlled whereas in the field, presence of other disease and microorganisms in the soil, cultivation and soil temperature and soil moisture vary considerably throughout the growing season.
Furthermore once a field-effective bacteria was selected, the problem of an economical and practical method of applying the bacteria in a commercial setting remained to be found.